3D Collagen Culture for Cell Imaging

Collagen gels were performed as previously described (Infante et al, 2018 (link)). Briefly, glass coverslips were layered with 15 μl of a 2.2 mg/ml type‐I collagen solution (bottom layer). Polymerization was induced at 37°C for 3 min. Then, a cell suspension (1.5–2.5 × 10⁵ cells/ml) was added to the bottom layer and cultures were incubated for 30 min at 37°C to allow cells to adhere to the collagen gels. Growth medium was gently removed and a 2.2 mg/ml type‐I collagen solution was polymerized on top of the cells (top layer). After polymerization at 37°C for 90 min, growth medium was added to the cultures. Z‐stacks of images were acquired with an inverted Nikon microscope coupled with a spinning disk confocal head (Andor) with a 60× objective.